Coriobacteriaceae modulated liver physiology

The liver is the central organ for metabolism of nutrients and metabolites absorbed in the intestine and transported via portal blood and for lipid, bile acid, and cholesterol homeostasis. The fact that Coriobacteriaceae modulate lipid homeostasis in WAT hints at possible effects on liver physiology.

Coriobacteriaceae are involved in the metabolism of bile acids, synthesized from cholesterol in the liver. Moreover, Martinez et al. (2013) showed that the occurrence of Coriobacteriaceae was associated with cholesterol absorption and synthesis [116]. Hence, we first analyzed in detail cholesterol homeostasis in the mice.

CORIO mice were characterized by cholesterol levels of approximately 150 to 210 µM in systemic plasma, which was significantly elevated compared to GF (ca. 1.5-fold) and SPF mice (ca. 2.5-fold) (Figure 18A). Strikingly, this CORIO-associated hypercholesterolemia was observed in all diet groups.

Levels in GF mice were generally 1.7-fold higher than in SPF, with concentrations of 55 to 205 µM depending on diet. Moreover, in agreement with the interesting WAT phenotype described above, higher levels of cholesterol were also observed in iWAT of BA-fed CORIO mice (CORIO, 110.6 ± 107.2 µg/ g iWAT; GF, 47.7 ± 33.1; SPF, 74.6 ± 49.7) (Figure 18B).

The observed systemic hypercholesterolemia raised the question whether CORIO mice have altered secretion, absorption or de novo synthesis of cholesterol. Therefore, cholesterol levels were measured in caecal content, but CORIO mice did not show any changes, regardless of the diet (Figure 18C). Interestingly, significantly higher cholesterol levels were observed in the caeca of GF mice fed P-HFD (2.6 ± 0.3 vs CORIO: 1.9 ± 0.7 and SPF: 1.7 ± 0.2 mg/ g content). In portal plasma, CD-fed CORIO mice had two-fold higher cholesterol levels compared to GF and SPF mice (Figure 18D). No CORIO-specific differences were found in the other two dietary groups in portal blood, indicating that cholesterol absorption was not increased in those mice. Accordingly, the mRNA expression of the ileal cholesterol transporter Niemann Pick C1 like protein 1 (NPC1L1) was not affected (Figure 18E).

Expression of the key enzyme for cholesterol de novo synthesis, HMG CoA reductase, did not show significant differences regarding colonization status or diets either (Figure 18F).

A

Figure 18: Coriobacteriaceae induced hypercholesterolemia.

(A) Measurement of systemic cholesterol levels revealed hypercholesterolemia in CORIO mice regardless of the diet. Cholesterol levels in iWAT (B), caecal content (C), and portal plasma (D). (E) Transcription analysis of the cholesterol transporter Npc1l1 in total ileal tissue. (F) Hepatic expression of Hmgcr, the key enzyme in cholesterol de novo synthesis. CD; P-HFD; BA; For detailed description of the statistical analysis see section 3.17; Number of mice in each group are indicated below the x-axis (n = number of mice measured).

A common liver dysfunction associated with dietary and microbial factors is non-alcoholic fatty liver disease (NAFLD), which is characterized by steatosis (fat accumulation), changes in bile acid composition, and is also associated with hypercholesterolemia. Therefore, Coriobacteriaceae might influence the development of NALFD via their metabolic functions.

Measurement of liver weight showed that P-HFD triggered a significant increase in all three colonization groups, accompanied by increased NAFLD activity score (Figure 19A and B). No differences in liver weight or NAFLD score were observed between CD and BA fed mice. Another marker for NAFLD is an increased hepatic triglyceride content. P-HFD-fed mice had 3.4- to 11.3-fold higher triglyceride load compared to CD- and BA-fed mice (Figure 19C). Interestingly, hepatic triglyceride content was higher in CORIO mice fed the BA diet compared to GF and SPF on the same diet (12.0 ± 4.3 vs. 10.2 ± 3.3 and 4.3 ± 3.2 mg/ g, respectively), but results did not reach significance in GF mice.

liver histopathological score (0-8) ***

Figure 19: P-HFD induced NAFLD and CORIO mice fed BA had higher hepatic triglyceride levels.

(A) Liver weight measurement after 21 weeks of feeding. (B) Histopathological evaluation and representative pictures for P-HFD and BA diet of H&E stained liver sections. (C) Hepatic triglyceride concentrations. CD; P-HFD;

BA; For detailed description of the statistical analysis see section 3.17; Number of mice in each group are indicated below the x-axis (n = number of mice measured).

This increase in hepatic triglycerides and the increased fat mass observed in BA-fed CORIO mice might be due to changes in the endogenous hepatic lipid metabolism (Figure 20A). Lipidomics of liver tissue revealed that BA-fed CORIO mice had a 1.6-fold higher amount of total fatty acids than SPF mice (152.6 ± 56 vs. 95.3 ± 26.5 nmol/ mg; p = 0.002), but differences in GF mice were not significant (131.2 ± 23.8 nmol/ mg; p = 0.383) (Figure 20B). Looking at fatty acid composition, a shift from

CORIO mice (Figure 20C). In line with this, CORIO mice had 2.5 to 12.6 % higher levels of C18:1(n-9) and 2.9 to 6.7 % lower levels of C18:2 (n-6) compared to GF and SPF mice (Figure 20D).

Next, we asked whether the observed different proportions of MUFAs were due to differences in fatty acid desaturation capacities. Therefore, the desaturation index was calculated for C16:0 and C18:0. In BA-fed CORIO mice, the C16:0 desaturation index was 1.3- to 2.1-fold higher compared to GF and SPF mice, respectively (Figure 20E). Furthermore, the C18:0 desaturation index was 2.2- to 2.6-fold higher in GF and CORIO than SPF mice. This increases desaturation capacity would imply a higher activity of hepatic SCD1, the key enzyme for fatty acid desaturation. However, measurement of hepatic Scd1 mRNA expression did not show any differences (Suppl. Fig. S5). As SCD1 is beside others regulated by SREBP1c, the mRNA expression of Srebf1c was measured. Interestingly, CORIO mice fed BA diet showed a 1.9-fold higher expression compared to SPF mice (Figure 20F).

A

hepatic fatty acid distribution

% of all detected fatty acids

GF(n=11)

hepatic total fatty acid composition

% of all detected fatty acids

GF (n=11)

ratio [sat. FA/ monounsat. FA]

GF (n=11)

hepatic total fatty acid concentration

sum of all detected lipids [nmol/mg]

GF(n=11)

ratio [sat. FA/ monounsat. FA]

GF (n=11)

Figure 20: Coriobacteriaceae modulated hepatic fatty acid amount and composition in BA-fed mice.

(A) Overview of endogenous fatty acid synthesis [248]. (B) Calculation of the total amounts of detected fatty acids (FA). (C) Amounts of total saturated (SA), monounsaturated (MU) and polyunsaturated fatty acids (PUFA).

(D) Hepatic total FA composition. Black boxes indicate significant differences. (E) Desaturation index, which is the ratio of monounsaturated to saturated FA, was used to analyze the activity of the hepatic enzyme Stearoyl-CoA desaturase (SCD1). (F) Hepatic expression of sterol regulatory binding protein 1c (Srebp1c), which controls fatty acid de novo synthesis. BA; For detailed description of the statistical analysis see section 3.17. Number of mice in each group are indicated in brackets (n = number of mice measured); FASN, fatty acid synthase; ELOVL, elongation of very long chain fatty acids protein

The lipidomics results aforementioned showed that Coriobacteriaceae interfere with hepatic lipid metabolism. The gut microbiota in SPF mice triggered down-regulation of the fatty acid transporter CD36 (also referred to as FAT) when fed CD and BA but not P-HFD diet. (Figure 21A). However, no Coriobacteriaceae-specific effects were observed. Interestingly, expression analysis of hepatic Pparγ, a key modulator of glucose and lipid homeostasis, revealed 2.7- to 6.9-fold higher levels in CORIO than GF and SPF mice fed BA diet (Figure 21B) Furthermore, Pparγ expression was 2.9- to 45-fold induced by P-HFD, depending on the colonization and diet with the highest induction in SPF mice.

Furthermore, the expression of Nr1h3, which codes for liver X receptor (LXR), a key regulator of lipid and cholesterol homeostasis, was reduced by 20 % in CORIO than SPF mice which is a minor but significant difference (Figure 21C).

A

Hepatic mRNA expression of Cd36 (A), a fatty acid transporter, and Pparγ (B), a modulator of lipid and glucose homeostasis as well as Nr1h3 (C), a regulator of bile acid, cholesterol and lipid metabolism CD; P-HFD; BA; For detailed description of the statistical analysis see section 3.17; Number of mice in each group are indicated below the x-axis (n = number of mice measured).

In summary, all CORIO mice were characterized by systemic hypercholesterolemia and increased cholesterol levels in iWAT when fed BA diet. Additionally, BA-fed CORIO mice had higher hepatic triglyceride levels, accompanied by increased fatty acids content and shifts in their composition.

4.2.6 Colonization with Coriobacteriaceae resulted in slight modulation of host bile acid